Vesicular Ca2+ Channels in Sarcolemma Repair and Muscular Dystrophy The mechanisms by which the plasma membrane maintains its integrity through active repair processes are poorly understood. Defects in plasma membrane repair have been linked to numerous disease conditions, including muscular dystrophy (MD), a group of inherited muscle diseases characterized by skeletal muscle wasting and weakness. Sarcolemma repair requires a rapid Ca2+ increase in order to trigger recruitment of intracellular vesicles to fuse with plasma membrane, replacing the disrupted membranes. The nature of the vesicles and the source of Ca2+ are still unclear. We have now identified a TRP-type Ca2+ channel in the lysosome as a core component of the membrane repair machinery. The primary observation that led to our discovery was that mice lacking the Mucolipin Transient Receptor Potential 1 (TRPML1) gene developed a primary, early onset, progressive muscular dystrophy, highly similar to the most common form (i.e., Duchenne) of Muscular Dystrophy caused by dystrophin mutations. Human mutations of TRPML1 cause Mucolipidosis type IV, a lysosome storage disease (LSD) characterized by motor impairment and mental retardation. By performing various sarcolemma repair assays using mouse knockouts and highly specific channel agonists/antagonists, we demonstrated in our preliminary studies that sarcolemma resealing is defective when TRPML1 is genetically inactivated or pharmacologically inhibited. The primary goal of this R21 application is to test the central hypothesis that TRPML1 promotes Ca2+-dependent lysosomal trafficking to reseal damaged sarcolemma. Aim 1 is to investigate the molecular mechanisms that link membrane damage to TRPML1 activation, vesicle trafficking, and lysosomal exocytosis in sarcolemma repair. We will use microelectrode penetration and membrane-disrupting toxins to cause muscle membrane damage/injury. We will then use electrophysiology to measure TRPML1 channel activity, Ca2+ imaging to measure lysosomal Ca2+ release, spinning-disk live imaging to monitor vesicle trafficking, and Lamp1 surface staining and lysosomal enzyme release to monitor lysosomal exocytosis. Aim 2 is to investigate the in vivo muscle protective effects of TRPML1 in the mouse models of muscular dystrophy. We will test whether small-molecule TRPML agonists can accelerate lysosomal trafficking to facilitate membrane repair and alleviate muscular dystrophy in mdx mice. Finally, we will investigate whether transgenic expression of TRPML1 in skeletal muscle can rescue the muscle weakness in mdx mice.